PROJECT SUMMARY. The heme nitric oxide and/or oxygen binding (H-NOX) proteins form a class of gas sensing proteins involved in binding gases and initiating signaling cascades in both prokaryotic and eukaryotic cells with a key member of this class being the NO-binding heme domain of soluble guanylate cyclase (sGC), the main NO sensor in humans. Unnatural amino acids (UAAs) are chemically synthesized molecules with minor alterations from naturally occurring amino acids that have been used to help study various biologically and medically relevant protein systems. UAAs have the distinct advantage of being chemically customizable for an application of interest and site-specifically incorporated into proteins while being minimally invasive of protein structure as the UAAs themselves are not much larger than native amino acids. This project utilizes the amber codon methodology to incorporate unnatural amino acids in H-NOX proteins to both study conformational changes and tune gas-binding affinity. The first aim of this project is to utilize vibrational reporter UAAs containing nitrile or azido groups to monitor solvation environments and conformational changes of two bacterial H-NOX proteins. The second aim of this project is to tune the oxygen-binding affinity of the O2-binding Thermoanaerobacter tencongensis H-NOX using UAAs. This research employs a number of biophysical techniques (FTIR, stopped-flow UV-Visible spectroscopy, laser flash photolysis, CD spectroscopy, and X-ray crystallography) to analyze the UAA-incorporated H-NOX mutants. These studies will extend our understanding of these important gas-binding proteins, work towards developing a new O2 delivery molecule with potential use as a therapeutic agent to treat hypoxia-related diseases, and expand the applications and utility of unnatural amino acids to study and/or engineer the structure and function of other biomedically relevant proteins.